The advantageous use of hypoxic tumour cells in cancer therapy: identical chemosensitization by metronidazole and misonidazole at moderately elevated temperatures.

Chemosensitization by two nitroimidazoles (NIs), metronidazole (METRO) and misonidazole (MISO), of the anti-tumour effect of alkylating agents was studied at three different temperatures: room temperature (RT), 37 and 41.5 degrees C. Three alkylating agents, cyclophosphamide (CY), 1,3 bis(2-chloroethyl)-N-nitrosourea (BCNU) and melphalan (L-PAM) were tested when the tumours reached an average diameter of 4 mm. Tumours were 4th generation isotransplants of a spontaneous fibrosarcoma, FSa-II. Treatment at 37 or 41.5 degrees C was given by immersing the tumour-bearing foot for 60 min in a water bath set at these temperatures. The test agents were injected ip immediately before immersing the foot in the water bath, whereas METRO or MISO (2.5 mmol/kg) was given ip 30 min before the injection of a test agent. Following treatment the tumour growth (TG) time, i.e. the time required for one-half of treated tumours to reach 1000 mm3 after the treatment day, was studied. For CY, MISO was a better sensitizer than METRO at RT and 37 degrees C, but the magnitude of the chemosensitization by MISO and METRO became identical at 41.5 degrees C. Notably, the chemosensitization was substantially enhanced at 41.5 degrees C, whereas neither 41.5 degrees C-heat, NIs or combined NI and heat prolonged the TG time. Although no chemosensitization was observed for BCNU at RT, both METRO and MISO equally enhanced the effect of BCNU at 41.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of radiation-induced chromosome damage and micronucleus induction in mouse bone marrow by misonidazole and hyperthermia.

The effect of misonidazole (MISO), local hyperthermia (HT) and their combination on radiation-induced chromosome damage and micronucleus (MN) induction was studied in mouse bone marrow cells. It was found that MISO treatment did not enhance the clastogenic effect of radiation, which indicates a lack of radiosensitization of bone marrow chromosomes. But post-irradiation HT increased the frequency of aberrant cells and MN. A combination of MISO and HT produced a significant increase in the frequency of radiation-induced aberrant cells and MN at all the radiation doses as compared to radiation alone. The percentage of aberrant cells as well as the percentage of MN showed a linear quadratic increase with radiation dose in all the treatment groups. At higher radiation doses, cells with > 1 MN increased quadratically with a pronounced increase in cells bearing > 2 MN and severely damaged cells (SDCs) at radiation doses above 3.0 Gy in the HT and MISO+HT treated groups. Our results indicate that though MISO itself may not have a radiosensitizing effect on mouse chromosomes, a combination of MISO with HT can enhance the radiation damage in normal bone marrow.

Free radical mechanism in enhancement of radiosensitization by SRSBR.

The free radical mechanism of enhancing radiosensibility by the Synergic Recipe to Strengthen Body Resistance (SRSBR) consisting of 10 Chinese drugs was studied by electron spin resonance (ESR) with spin trapping reagent--Nitroso-tert-butane (NtB) following irradiation of the Deoxy thymidine (dT) solution system by 60Co 3.7 PBq. The results showed SRSBR cannot only enhance the generation of e- aq but also enhance the production of . OH and H . after irradiation with gamma ray in dT-NtB-SRSBR aqueous solution system. These result in damage to biological molecules, attacking and killing tumor cells radiosensitized by SRSBR. The characteristics of SRSBR for radiosensitization are concluded to be increasing production of . OH and H. in comparison with the well-known radiosensitizer-Misonidazole (Miso).

Effect of hyperthermia on the activity of 1-[(4'-hydroxy-2'-butenoxy)methyl]-2-nitroimidazole, which is cytotoxic to hypoxic cells.

The effect on EMT6/KU cells of a newly synthesized hypoxic cell sensitizer, 1-[(4'-hydroxy-2'-butenoxy)methyl]-2-nitroimidazole (RK28), combined with heat was determined in vitro under conditions of hypoxia. As compared with aerobic conditions, hypoxia produced a 1.30-fold increase in the cytotoxicity of the drug for mouse mammary EMT6/KU cells induced by 1 h heat treatment at 43 degrees C in medium with a normal pH. Hypoxia also reduced the surviving fraction of cells treated with both RK28 alone for 2 h and the same concentrations of RK28 and heat (43 degrees C) in combination. Those enhancement ratios corresponded to a 20.3- and > 345-fold increase, respectively. Moreover, concomitant treatment with RK28 and heat greatly inhibited the clonogenic activity of the EMT6/KU cells under conditions of in vitro hypoxia and in all experimental groups; there was a statistically significant difference in the time-response curves (P < 0.05). As hypoxic cells in a solid tumor are resistant to various anticancer drugs, RK28 combined with hyperthermia deserves further study for possible clinical applications.

Effect of oxygenation, pH and hyperthermia on RSU-1069 in vitro and in vivo with radiation in the FSaIIC murine fibrosarcoma.

We have evaluated the combination of the radiosensitizing and bioreductive alkylating agent RSU-1069 with hyperthermia and radiation in an attempt to improve the potential effectiveness of hyperthermia and radiation against locally advanced malignancies. In vitro studies in FSaIIC murine fibrosarcoma cells demonstrated that 1 h exposure to RSU-1069 was more cytotoxic toward hypoxic than normally oxygenated cells at 37 degrees C and pH 7.40 and was only minimally more cytotoxic at hyperthermic temperatures. At pH 6.45, however, RSU-1069 became significantly more toxic toward hypoxic cells and its cytotoxicity was markedly increased at hyperthermic temperatures. In contrast, the ability of this agent to radiosensitize hypoxic FSaIIC cells significantly diminished at pH 6.45. Hoechst 33342 diffusion selected FSaIIC tumor subpopulation studies revealed that hyperthermia and RSU-1069 were more toxic towards dim (hypoxic) cells, while radiation was more toxic towards bright (normally oxygenated) cells. A combination of all three modalities resulted in an equal and significant kill of hypoxic and oxygenated cells. These results suggest that the combination of RSU-1069, local hyperthermia and radiation has considerable clinical potential.

Evaluation of nitroimidazole hypoxic cell radiosensitizers in a human tumor cell line high in intracellular glutathione.

Five nitroimidazole hypoxic cell radiosensitizers were evaluated in a human lung adenocarcinoma cell line (A549) whose GSH level was 8-fold higher than Chinese hamster V79 cells. One millimolar concentrations of Misonidazole (MISO), SR-2508, RSU-1164, RSU-1172, and Ro-03-8799 sensitized hypoxic A549 cells to radiation, with Ro-03-8799 giving the highest sensitizer enhancement ration (SER) (2.3). However, MISO, SR-2508 and Ro-03-8799 were less effective in this cell line than in V79 cells, presumably due to higher GSH content of the A549 cells. Increased hypoxic radiosensitization was seen with 0.1 mM Ro-03-8799 after GSH depletion by BSO as compared to 0.1 mM Ro-03-8799 alone (SER-1.8 vs 1.3). The combination of GSH depletion and 0.1 mM Ro-03-8799 was considerably more toxic than 0.1 mM or 1.0 mM Ro-03-8799 alone. This sensitivity was much greater than has been observed for SR-2508. These data show that Ro-03-8799 was the most efficient hypoxic cell radiosensitizer in a human tumor cell line considerably higher in GSH than the rodent cell lines often used in hypoxic radiosensitization studies. Thus, Ro-03-8799 may be a more effective hypoxic cell sensitizer in human tumors that are high in GSH.

Misonidazole-induced radio-resistance in normal and preirradiated jejunal mucosa.

Pretreatment of mice with a single dose of whole-abdomen irradiation led to a relative decrease in radiosensitivity in jejunal crypts given a second single dose of irradiation 2 months later. Hyperbaric oxygen (3.5 bars) restored the survival level to initial values, suggesting that there was radiation-induced hypoxia in the primed jejunum. However, misonidazole did not sensitize primed jejunal crypts; it reduced the radiosensitivity of both normal and primed crypts. This 'paradoxical' effect of misonidazole could well be due to the acute toxic side-effects of 1 mg/g body weight misonidazole i.p., as there was a sharp drop in the core temperature of mice immediately after drug injection. Artificially maintaining the temperature of miso-treated animals at a normal level produced crypt survival levels similar to those of both normal controls and primed controls. Thus, although the primed gut is chronically hypoxic, as suggested by the effects of hyperbaric oxygen, misonidazole is not a reliable tool for the study of this tissue hypoxia. In all in vivo experiments with misonidazole, core temperature must be controlled in order to avoid misleading interpretations of experimental results.

The effect of previous treatment on the response of mouse feet to irradiation and hyperthermia.

The response of mouse feet to irradiation and heat was studied 90 days after a first treatment with X-rays, hyperthermia or both. Residual damage after a single dose of 20-30 Gy enhanced both the acute reaction and "late" deformity following a second treatment with radiation or hyperthermia. There was often a larger "memory" of the first radiation treatment for late deformity compared with the acute skin response, especially in the case of retreatment by hyperthermia. Prior treatment of the foot with a moderate heat dose (60 min at 44 degrees C), which by itself did not lead to deformity, had only a small effect on the response to retreatment with irradiation or heat, both with respect to the acute and "late" response. Residual damage after more severe hyperthermia (90 min at 44 degrees C) obscured the evaluation of deformity after a second treatment with radiation or hyperthermia. Feet treated with irradiation followed immediately or after 3 days by heat, show a larger "memory" when retreated with hyperthermia than with irradiation, both with regard to the acute and "late" response. Experiments using misonidazole indicated that the oxygenation status of previously treated skin (pretreatment not leading to deformity) had not changed significantly.

Enhancement of misonidazole chemopotentiation by mild hyperthermia (41 degrees C) in vitro and selective enhancement in vivo.

Experiments were designed to test the hypothesis that mild heat treatment would selectively increase misonidazole (MISO) chemopotentiation of CCNU toxicity in hypoxic versus aerobic cells in vitro and in tumours in vivo via an augmentation of nitroreduction. EMT-6 cells were exposed to CCNU +/- 1.0 mM MISO under aerobic or hypoxic conditions for 4 h either at a constant 37 degrees C or at 41 degrees C for the first hour followed by 37 degrees C for the remaining 3 h. Chemopotentiation was not observed under aerobic conditions and heat treatment did not modify CCNU toxicity. Co-incubation with MISO and CCNU under hypoxic conditions resulted in enhanced toxicity (i.e. chemopotentiation) with either incubation protocol; however, the magnitude of the enhancement was significantly larger (P less than 0.025) when 41 degrees C incubation was included. Systemic heat treatment produced a similar enhancement of chemopotentiation in KHT tumours in C3H/HeN mice treated with MISO (0.5 mg g-1) and whole body hyperthermia (41 degrees C, 1 h) prior to administration of CCNU (15 mg kg-1). Heating had no effect on CCNU response but doubled the median growth delay produced by the CCNU-MISO combination. Heat treatment did not enhance myelosuppression of the combination. Both the in vitro and in vivo data indicate that mild hyperthermia can selectively enhance the magnitude of MISO chemopotentiation.

An in vitro and in vivo screening system for new hypoxic cell radiosensitizers using EMT6 cells.

An experimental tumor system consisting of single cells, spheroids, and solid tumors was developed using the EMT6/KU cell-line for evaluation of new hypoxic cell sensitizers. This paper describes the radiation dose-survival curves of the system and the effects of four sensitizers [misonidazole, Ro 03-8799, KB-11 (a sulfonyltetrazole derivative), and DNIE (a dinitroimidazole derivative)] in this system. Dose-survival curves of the spheroids varied significantly depending on the irradiation conditions, but it was found that the spheroids could model the solid tumors when they were irradiated in flat-bottomed flasks. These spheroids and solid tumors contained substantial fractions of hypoxic cells, and hence were suitable for testing hypoxic cell sensitizers. Misonidazole at a concentration of 1 mM or 1 mmol/kg showed a constant enhancement ratio (ER) of 1.55 in all of the systems. The ERs of the other three compounds for single cells, spheroids, and solid tumors were 2.1, 1.9, and 1.65, respectively, for Ro 03-8799 (1 mM or 1 mmol/kg); 2.6, 1.6, and 1.0, respectively, for KB-11 (0.5mM or 0.5 mmol/kg); 1.8, 1.2, and 1.1, respectively, for DNIE (0.5mM or 0.5 mmol/kg). These results indicated that the ER for spheroids is closer to the ER for solid tumors than is the ER for single cells in most cases. It was also suggested that an efficient sensitizer in vivo shows little or no difference between its ER for single cells and that for spheroids.

Inhibition of glutathione peroxidase and glutathione transferase in mouse liver by misonidazole.

The mechanisms of toxicity and sensitization by the radiosensitizer misonidazole [1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol] are not well understood. We report here on the inhibition of total glutathione peroxidase (GSHPx), selenium-dependent glutathione peroxidase (selenium-GSHPx) and glutathione transferase (GSHTx) activities by misonidazole. Mouse liver cytosol GSHPx and selenium-GSHPx were inhibited in vitro with 0.5 mM misonidazole. On administration of the drug intraperitoneally (800 mg/kg) to mice, it was found that GSHPx, selenium-GSHPx, and GSHTx were inhibited in homogenate, cytosol, and microsomal fractions of mouse liver. GSHPx was depressed in all fractions up to 60-70% of control values, with maximum depression occurring in the cytosol and homogenate fractions in less than 2 hr. Recovery of activity was slower in the microsomes. In general, the pattern of depression of selenium-GSHPx was parallel to that of GSHPx except in microsomes, where GSHPx is minimal. Quantitatively, selenium-GSHPx was least affected. GSHTx was inhibited 70-80% of control values in cytosol and homogenate with recovery by 24 hr, whereas a second period of depression occurred at 24 hr in the microsomes. The inhibition of peroxide-metabolizing enzymes may lead to elevation of intracellular peroxide levels, contributing to the radiosensitizing effect and/or toxicity of misonidazole.

[Radiosensitization research in cancer therapy].

Radiosensitization using Synkavit was first reported by Mitchell in 1953. Recently, renewed interest in radiosensitization has been shown by tumor radiobiologists since electron-affinitive hypoxic cell sensitizers were introduced Adams and his colleagues in 1973. Conferences on chemical modifiers i.e., radiosensitizers and radioprotectors, have been held every two years since 1977 in Britain or North America. At the last meeting in Banff, Canada in 1983 the results of randomized clinical trials of misonidazole were found to be rather disappointing and non-hypoxic cell sensitizers such as halogenated thymine analogues and PLD repair inhibitors were introduced. In parallel with these approaches, hyperthermia research combined with radiation was started in 1974. Very effective radiosensitization by heat-treatment, for example 43 degrees C for 40 min, has been shown in in vitro as well as in vivo experiments. Enhancement of the anti-tumor activity of some chemotherapy drugs using hypoxic cell sensitizers or PLD repair inhibitors was found to be a new approach for improving cancer chemotherapy in 1982. Hyperthermia was also shown to enhance the anti-tumor activity of some chemicals. Thus radiosensitization research may be extended to chemosensitization. i.e., from selective sensitization used in local radiotherapy to that used in systemic chemotherapy.

The effect of hypoxic radiosensitizer after mild hyperthermia in C3H mammary carcinoma.

The radiosensitizing effect of misonidazole after hyperthermia was investigated in C3H mammary carcinoma. The tumors transplanted into the flanks of the mice were heated in a 42.3 degrees C water bath for 30 min. When misonidazole was administrated before heating, the subsequent radiation effect was prominently enhanced, whereas post-heating administration of misonidazole did not enhance the radiation effect significantly. The effect of varying the time between heating and radiation with or without misonidazole was as follows. Without misonidazole, the radiation effect was decreased at 6 hours after heating but increased at 12 hours, then it returned to the initial level at 24 hours and remained until 96 hours after heating. With misonidazole administered 30 min before irradiation, the radiosensitizing effect was observed at 24, 48 and 96 hours after heating. However, the total effects of this procedure were almost the same as the results in the combination without heating. Changes in the hypoxic fraction after hyperthermia are also discussed.

Clinical trials of radiosensitizers: what should we expect?

The lack of positive results from the clinical trials undertaken so far with misonidazole (MISO) are widely considered as disappointing. This is leading to a growing sentiment that hypoxic cells may not be a significant limitation to local control of human tumors. To examine whether this is a reasonable conclusion, the relevant in vitro and in vivo data have been summarized so that predictions of the extent of radiosensitization of the hypoxic cells can be made from a knowledge of the clinically achievable levels of MISO. This analysis shows the following: First, the original curve of Adams with V-79 cells is probably over-optimistic in predicting sensitizer enhancement ratios (SERs). A new curve based on the available in vivo data predicts lower sensitization so that even at the highest MISO doses used clinically, SERs for the hypoxic cells to large single X-ray doses of only 1.45 would be expected. In a clinical trial, reoxygenation of the hypoxic cells is likely to occur, thereby considerably reducing the SER for the total tumor cell population. This, together with the problems of heterogeneous tumors and insufficient patient numbers, could well have been responsible for the negative clinical results. Second, even if tumor levels of the new radiosensitizer SR-2508 10 times those of MISO can be achieved clinically, this will still not lead to full radiosensitization of the hypoxic cells (although an SER in excess of 2.0 should be attainable). In conclusion the in vitro and in vivo data with radiosensitizers suggest that only a small effect, if any, is likely to be demonstrated in the clinical trials with MISO, even for those tumors the control of which is limited by hypoxic cells. Thus the question of whether hypoxic cells may or may not limit the local control of tumors by radiotherapy has not been addressed adequately by the presently available radiosensitizing drugs.

In vivo testing of a 2-nitroimidazole radiosensitizer (Ro 03-8799) using repeated administration.

The radiosensitizing efficiency of a novel 2-nitroimidazole (Ro 03-8799) has been compared with that of misonidazole. Each compound was assessed by constructing dose response curves for regrowth delay using a range of drug doses. The concentration of each compound was measured in tumor homogenates with high performance liquid chromatography (HPLC). When compared on the basis of administered dose the new compound was no more efficient than misonidazole. Comparison on the basis of measured tumor concentrations showed that Ro 03-8799 was 3--4 times more efficient than misonidazole, but only at very high drug levels. Previous in vitro studies had shown a constant 10-fold difference in potency. In order to eliminate possible artifacts caused by the short half life in mice, repeated injections of Ro 03-8799 were used to maintain a constant tumor concentration for two hours before irradiation. No increase in effectiveness was observed with prolonged exposure. This is a charged compound whose distribution is pH dependent; gross tumor levels should therefore be interpreted with caution.

Use of solubilizing agents to study radiosensitization in vitro by compounds of low water solubility.

A major disadvantage in studying the effects of chemicals on the radiation response of mammalian cells in vitro can be the poor water solubility of the chemicals. Using Chinese hamster cells we have overcome this problem by using dimethyl sulphoxide and ethanol as co-solvents. From studies using these co-solvents and misonidazole as a standard we have demonstrated that these co-solvents have no effect on sensitizing efficiency and cytotoxicity. A number of poorly water soluble 2-nitroimidazoles of promising structure have been studied. The results, together with those from studies on misonidazole and the co-solvents are presented.